Infections of domestic and wild birds with low-pathogenic avian influenza viruses (LPAIVs) have been associated with protective immunity to subsequent infection. However, the degree and duration of immunity in wild birds from previous LPAIV infection, by the same or a different subtype, are poorly understood. Therefore, we inoculated H13N2 (A/black-headed gull/Netherlands/7/2009) and H16N3 (A/black-headed gull/Netherlands/26/2009) LPAIVs into black-headed gulls (Chroicocephalus ridibundus), their natural host species, and measured the long-term immune response and protection against one or two reinfections over a period of > 1 year. This is the typical interval between LPAIV epizootics in wild birds. Reinfection with the same virus resulted in progressively less virus excretion, with complete abrogation of virus excretion after two infections for H13 but not H16. However, reinfection with the other virus affected neither the level nor duration of virus excretion. Virus excretion by immunologically naive birds did not differ in total levels of excreted H13 or H16 virus between first-and second-year birds, but the duration of H13 excretion was shorter for second-year birds. Furthermore, serum antibody levels did not correlate with protection against LPAIV infection. LPAIV-infected gulls showed no clinical signs of disease. These results imply that the epidemiological cycles of H13 and H16 in black-headed gulls are relatively independent from each other and depend mainly on infection of first-year birds. IMPORTANCE Low-pathogenic avian influenza viruses (LPAIVs) circulate mainly in wild water birds but are occasionally transmitted to other species, including humans, where they cause subclinical to fatal disease. To date, the effect of LPAIV-specific immunity on the epidemiology of LPAIV in wild birds is poorly understood. In this study, we investigated the effect of H13 and H16 LPAIV infection in black-headed gulls on susceptibility and virus excretion of subsequent infection with the same or the other virus within the same breeding season and between breeding seasons. These are the only two LPAIV hemagglutinin subtypes predominating in this species. The findings suggest that H13 and H16 LPAIV cycles in black-headed gull populations are independent of each other, indicate the importance of first-year birds in LPAIV epidemiology, and emphasize the need for alternatives to avian influenza virus (AIV)-specific serum antibodies as evidence of past LPAIV infection and correlates of protection against LPAIV infection in wild birds.